The long-term goal of our research is to understand the roles of axon guidance molecule Robo4 signaling and heparan sulfate (HS) proteoglycans in vasculature and gain new knowledge to guide development of Robo4- and HS-based novel therapeutics for human diseases. Our recent studies uncovered that Slit3 interacts with Robo4 to promote endothelial cell (EC) functions in vitro and neovascularization in vivo, and demonstrated that EC-HS promotes developmental angiogenesis in diaphragm by functioning as a co- receptor for Slit3-Robo4 signaling. These studies documented in vivo evidence that EC-HS is required for developmental angiogenesis in diaphragm and elucidated a positive regulatory role of EC-HS in Robo4 signaling. In our ongoing studies, we uncovered that Robo4 and syndecan-1 (SDC-1), the HS proteoglycan that functions as a co-preceptor by forming SDC-1-Slit3-Robo4 ternary complex on the EC surface, both are constitutively shed from the EC surface, and the shedding reduces cell surface levels of Robo4 and SDC-1 and attenuates Slit3-induced EC migration. These new findings revealed a shedding-based regulatory mechanism in Robo4 signaling and led to our first hypothesis that ectodomain shedding of Robo4 and SDC-1 both negatively regulates Robo4 signaling. In parallel, our new studies also generated and examined the EC- specific Ext1 knockout (Ext1ECKO) mice in which the expression of EC-HS is completely abolished. The Ext1ECKO mice exhibit profound and severe developmental angiogenesis defects and are embryonic lethal, revealing that EC-HS is essentially required for developmental angiogenesis in multiple organs. Phenotype characterization uncovered that the EC-Ext1 ablation disturbs EC functions, including tip cell sprouting and filopodia formation, and mural cell (MC) recruitment, phenocopying the vascular development defects displayed in mice that are deficient in vascular endothelial growth factor (VEGF) and platelet-derived growth factor-B (PDGF-B), respectively, leading to our second hypothesis that EC-HS facilitates both VEGF- and PDGF-B signaling to essentially modulate developmental angiogenesis. To vigorously test our new hypotheses we will pursue three Specific Aims. Aim 1 will delineate the regulatory roles of Robo4- and SDC- 1 ectodomain shedding on Robo4 signaling in angiogenesis by identifying Robo4- and SDC-1 sheddaes(s) and assessing if alteration of Robo4- and SDC-1 shedding affects Robo4 signaling. Aim 2 will determine the essential role of EC-HS in developmental angiogenesis and the underlying cellular mechanisms by characterizing vascular defects in the Ext1ECKO mice and assessing related EC- and MC functions. Aim 3 will decipher the molecular signaling, i.e., VEGF- and PDGF-B signaling by which EC-HS modulates developmental angiogenesis. The proposed studies will use both novel and established genetic, cellular and biochemical approaches in conjunction with in vitro and in vivo angiogenesis models. These serial investigations are anticipated to elucidate Robo4- and SDC-1 ectodomain shedding as a novel regulatory mechanism of Robo4 signaling in angiogenesis, to establish that EC-HS is essentially required for developmental angiogenesis, and to reveal that EC-HS facilitates both VEGF and PDGF-B signaling as the molecular mechanisms underlying the essential pro-angiogenesis function of EC-HS in development.